JP4857754B2 - Method for producing sulfur-containing hydroxycarboxylic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a sulfur-containing &alpha;-hydroxycarboxylic acid compound without the formation of a large amount of by-products or inhibition of an enzyme activity. <P>SOLUTION: The method for producing the sulfur-containing &alpha;-hydroxycarboxylic acid compound represented by formula (2) (wherein, R<SP>1</SP>is a hydrogen, a 1-8C alkyl group or a 6-20C aryl group) is carried out as follows. Rhodococcus rhodochrous or a microbial cell of a microorganism belonging to the Rhodococcus sp. or a treated material of the microbial cell having an ability to convert a sulfur-containing dihydroxy compound represented by formula (1) äwherein, R<SP>1</SP>is the same as in formula (2)} into the corresponding &alpha;-hydroxycarboxylic acid compound and cultured in the presence of a primary or a secondary alcohol is made to act on the sulfur-containing dihydroxy compound. <P>COPYRIGHT: (C)2007,JPO&amp;INPIT

Description

  The present invention relates to a method for producing a sulfur-containing hydroxycarboxylic acid.

Conventionally, in order to produce a sulfur-containing hydroxycarboxylic acid, a method of hydrolyzing cyanohydrin has been used. Industrially, a method using sulfuric acid as a catalyst is known. Also known are methods for hydrolyzing hydroxynitrile compounds by the action of microorganisms to convert them into the corresponding hydroxycarboxylic acids (see, for example, Patent Document 1, Patent Document 2, and Patent Document 3).
However, in the method using sulfuric acid as a catalyst, as a result of the reaction between the hydroxynitrile compound and sulfuric acid, the target product, hydroxycarboxylic acid, and an equimolar amount of ammonium sulfate are by-produced. In the method for producing a corresponding hydroxycarboxylic acid compound from a hydroxynitrile compound using microorganisms, the enzyme activity retained by the microorganisms due to cyanide or the like, which is a decomposition product from the hydroxynitrile compounds, is required. There is a problem that it is obstructed or a treatment of a large amount of ammonium salt to be produced is necessary, which is accompanied by an increase in production cost.

Japanese Patent Publication No.58-15120 Japanese Patent Laid-Open No. 2-84198 Japanese Patent Laid-Open No. 4-40898

  Provided is a process for producing a sulfur-containing hydroxycarboxylic acid which does not cause the production of a large amount of by-products and the inhibition of enzyme activity.

That is, the present invention
1. Formula (1)

（式中、Ｒ^１は水素、炭素数１−８のアルキル基、または、炭素数６−２０のアリール基を表す。）
で示される含硫ジヒドロキシ化合物に、当該含硫ジヒドロキシ化合物を対応するα−ヒドロキシカルボン酸化合物に変換する能力を有する、１級または２級のアルコール存在下で培養したロドコッカス・ロドクラウス（Rhodococcus rhodochrous）、または、ロドコッカス・エスピー（Rhodococcus sp.）に属する微生物の菌体又は菌体処理物を作用させることを特徴とする、式（２）

(Wherein R ¹ represents hydrogen, an alkyl group having 1 to 8 carbon atoms, or an aryl group having 6 to 20 carbon atoms.)
Rhodococcus rhodochrous cultured in the presence of a primary or secondary alcohol having the ability to convert the sulfur-containing dihydroxy compound into the corresponding α-hydroxycarboxylic acid compound, Alternatively, the microorganism (Rhodococcus sp.) Belonging to Rhodococcus sp. Is allowed to act, or a treated product of the microorganism (2)

（式中、Ｒ^１は前記と同じ意味を表す。）
で示される含硫α-ヒドロキシカルボン酸化合物の製造法（以下、本発明製造法と記すこともある。）；
２．前記微生物が、ロドコッカス・ロドクラウス（Rhodococcus rhodochrous）ATCC15610株、または、ロドコッカス・エスピー（Rhodococcus sp.）ATCC19148株の微生物である前項１記載の製造法；
３．式（１）で示される含硫ジヒドロキシ化合物におけるＲ^１が炭素数１−８アルキル基である前項１又は２記載の製造法；
４．１級または２級のアルコールが１級または２級の炭素数１〜５のアルコールである前項１〜３記載の製造法；
５．１級または２級のアルコールがメタノール、エタノール、１-プロパノール、２-プロパノール、１−ブタノール、２−メチル−１−プロパノール、２，２−ジメチル−１−プロパノールである前項１〜３記載の製造法；
等を提供するものである。

(Wherein R ¹ represents the same meaning as described above.)
A process for producing a sulfur-containing α-hydroxycarboxylic acid compound represented by the formula (hereinafter sometimes referred to as the process of the present invention);
2. The production method according to the above item 1, wherein the microorganism is a microorganism of Rhodococcus rhodochrous ATCC15610 strain or Rhodococcus sp. ATCC19148 strain;
3. The production method according to item 1 or 2, wherein R ¹ in the sulfur-containing dihydroxy compound represented by formula (1) is a C 1-8 alkyl group;
4. The production method according to items 1 to 3, wherein the primary or secondary alcohol is a primary or secondary alcohol having 1 to 5 carbon atoms;
5. The items 1 to 3 above, wherein the primary or secondary alcohol is methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-methyl-1-propanol, or 2,2-dimethyl-1-propanol. Manufacturing method of
Etc. are provided.

  According to the present invention, a sulfur-containing hydroxycarboxylic acid compound can be produced effectively without fear of the production of a large amount of by-products and the inhibition of enzyme activity.

Hereinafter, the production method of the present invention will be described.
In the sulfur-containing dihydroxy compound represented by the formula (1) and the sulfur-containing hydroxycarboxylic acid compound represented by the formula (2), the C 1-8 alkyl group represented by R ¹ includes, for example, a methyl group, ethyl Group, propyl group, isopropyl group, butyl group, isobutyl group, t-butyl group, pentyl group, hexyl group, heptyl group and octyl group. The aryl group having a carbon number of 6-20 represented by R ^1, for example, a phenyl group, a tolyl group, a xylyl group, a naphthyl group.
R ₁ in the sulfur-containing dihydroxy compound represented by the formula (1) is preferably an alkyl group having 1 to 8 carbon atoms.
Incidentally, the sulfur-containing hydroxycarboxylic acid compound represented by the formula (2) corresponding to the sulfur-containing dihydroxy compound represented by the formula (1), which is produced by the production method of the present invention and recovered from the reaction solution, is in the form of a salt. May be.
The compound of the formula (1) can also be produced, for example, according to the method described in EP1260500 or J. Agri. Food Chem. (1975) 1137 or a modification thereof.

  Microorganisms such as Rhodococcus rhodochrous and Rhodococcus sp. Having the ability to convert sulfur-containing dihydroxy compounds used in the production method of the present invention into the corresponding α-hydroxycarboxylic acid Examples of microbial cells or processed microbial cells cultured under the conditions include, for example, Rhodococcus rhodochrous ATCC15610 and Rhodococcus sp having the ability to convert a sulfur-containing dihydroxy compound into the corresponding α-hydroxycarboxylic acid. .) A microorganism of ATCC 19148, which is a microbial cell cultured in the presence of water or a treated microbial cell.

  According to the method of the present invention, the primary hydroxyl group of the sulfur-containing dihydroxy compound represented by the formula (1) can be preferentially oxidized. Here, “preferentially oxidizable” means that oxidation of the primary hydroxyl group proceeds preferentially over oxidation of the secondary hydroxyl group or sulfide oxidation of the sulfur-containing dihydroxy compound.

  Microorganisms such as Rhodococcus rhodochrous and Rhodococcus sp. Used in the production method of the present invention cultivate various microorganisms appropriately containing a carbon source, nitrogen source, organic salt, inorganic salt, and the like. The culture medium may be used for culture.

  Examples of the carbon source contained in the medium include glucose, sucrose, glycerol, starch, alcohol, organic acid and molasses. As the alcohols, primary or secondary alcohols having 1 to 5 carbon atoms are particularly preferable. For example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-methyl-1-propanol, 2,2-dimethyl-1-propanol is mentioned. Reactivity can be enhanced by culturing the microorganism in a medium to which alcohol has been added. Nitrogen sources include, for example, yeast extract, meat extract, peptone, casamino acid, malt extract, soy flour, corn steep liquor, cottonseed flour, dry yeast, ammonium sulfate and sodium nitrate, organic Examples of the salt and inorganic salt include sodium chloride, potassium chloride, sodium carbonate, 1 potassium phosphate, 2 potassium phosphate, calcium carbonate, ammonium acetate, magnesium sulfate, copper sulfate, zinc sulfate, ferrous sulfate and cobalt chloride. Is mentioned.

Examples of the culture method include solid culture and liquid culture (test tube culture, flask culture, jar fermenter culture, etc.).
The culture temperature and the pH of the culture solution are not particularly limited as long as the microorganism grows. For example, the culture temperature is in the range of about 15 to 45 ° C., and the pH of the culture solution is about 4 to 8. A range can be mentioned. The culture time can be appropriately selected depending on the culture conditions, but is usually about 1 to 7 days.

  The cultured cells can be used in the production method of the present invention as they are. As a method of using the cells of this microorganism as they are, (1) a method of using the culture solution as it is, (2) collecting the cells by centrifugation of the culture solution, and collecting the collected cells (if necessary, a buffer solution Or a method using a wet cell after washing with water.

  In the production method of the present invention, the processed microbial cell product obtained as described above can also be used. Examples of the treated cells include cells obtained by culturing cells treated with an organic solvent (acetone, ethanol, etc.), freeze-dried or alkali-treated, or cells physically or Enzymatically crushed or crude enzyme separated and extracted from these can be mentioned. Furthermore, what was fixed by the well-known method is included in a microbial cell processed material after giving the said process.

The production method of the present invention is usually performed in the presence of water. The water in this case may be in the form of a buffer solution. Examples of the buffer used in the buffer include alkali metal salts of phosphoric acid such as sodium phosphate and potassium phosphate, and alkali metal salts of acetic acid such as sodium acetate and potassium acetate.
In addition, the production method of the present invention can be carried out using a hydrophobic organic solvent in the presence of water and a hydrophobic organic solvent. Examples of the hydrophobic organic solvent used in this case include ethyl formate, ethyl acetate, propyl acetate, butyl acetate, ethyl propionate, butyl propionate, and the like, n-butyl alcohol, n-amyl alcohol, n- Alcohols such as octyl alcohol, aromatic hydrocarbons such as benzene, toluene and xylene, ethers such as diethyl ether, diisopropyl ether and methyl-t-butyl ether, and halogenated hydrocarbons such as chloroform and 1,2-dichloroethane And mixtures thereof.
Moreover, this invention manufacturing method can also be performed in presence of water and a hydrophilic organic solvent using a hydrophilic organic solvent. Examples of the hydrophilic organic solvent used in this case include alcohols such as methanol and ethanol, ketones such as acetone, ethers such as dimethoxyethane, tetrahydrofuran and dioxane, and mixtures thereof.

  The production method of the present invention is usually carried out within a pH range of 3 to 10 in the aqueous layer, but may be appropriately changed within the range in which the reaction proceeds.

  The production method of the present invention is usually performed within a range of about 0 to 60 ° C., but may be appropriately changed within a range in which the reaction proceeds.

  The production method of the present invention is usually performed within a range of about 0.5 hours to about 10 days. The end point of the reaction can be confirmed by measuring the amount of the sulfur-containing dihydroxy compound in the reaction solution, for example, by liquid chromatography, gas chromatography or the like after the addition of the sulfur-containing dihydroxy compound as the raw material compound. it can.

  The concentration of the sulfur-containing dihydroxy compound that is the raw material compound in the production method of the present invention is usually 50% (w / v) or less, and in order to keep the concentration of the sulfur-containing dihydroxy compound in the reaction system substantially constant, The sulfur-containing dihydroxy compound may be continuously or sequentially added to the reaction system.

  In the production method of the present invention, for example, a sugar such as glucose, sucrose, or fructose, or a surfactant such as Triton X-100 (registered trademark) or Tween 60 (registered trademark) is added to the reaction system as necessary. You can also.

  After completion of the reaction, the sulfur-containing hydroxycarboxylic acid compound corresponding to the sulfur-containing dihydroxy compound may be recovered from the reaction solution by subjecting the reaction solution to usual post-treatment such as organic solvent extraction, filtration, and concentration. The recovered sulfur-containing hydroxycarboxylic acid compound can be further purified by column chromatography, distillation or the like, if necessary.

EXAMPLES Hereinafter, although the Example manufacturing method is demonstrated in detail by an Example, this invention manufacturing method is not limited to these examples.

Synthesis Example of 4- (Methylthio) butane-1,2-diol To a 100 mL flask equipped with a magnetic rotator, 880 mg of 3-butene-1,2-diol and 10 mg of azobisisobutyronitrile were added. Methanethiol was bubbled through the mixture for 1 hour while stirring the mixture at 25 ° C. Further, the mixture was stirred at the same temperature for 1 hour, and then nitrogen was bubbled through the mixture for 0.5 hour to remove the remaining methanethiol, and finally 1245 mg of colorless oil was obtained. When this oil was analyzed by a gas chromatography area percentage method, the yield of 4- (methylthio) butane-1,2-diol was 72.5%. The amount of 3-butene-1,2-diol remaining unreacted was 26.6% of the charged amount.
Next, water was added to the obtained colorless oil so that the concentration of 4- (methylthio) butane-1,2-diol was 10% (w / v), and then insoluble matter (that is, azo By removing the bisisobutyronitrile by filtration, the raw material used in Example 1 or Example 2 (10% (w / v) 4- (methylthio) butane-1,2-diol aqueous solution) Got.

Example 1 (Production example of sulfur-containing hydroxycarboxylic acid compound from sulfur-containing dihydroxy compound by the production method of the present invention)
Sterilized medium (1 g of water, 20 g of various alcohols shown in Table 1, 5 g of polypeptone, 3 g of yeast extract, 3 g of meat extract, 0.2 g of ammonium sulfate, 1 g of potassium dihydrogen phosphate and 7 water of magnesium sulfate in a test tube) After adding 0.5 g of the Japanese product, the pH was adjusted to 7.0) 5 ml), and Rhodococcus rhodochrous ATCC15610 strain was inoculated therein. This was cultured with shaking at 30 ° C. under aerobic conditions. After culturing, the cells were separated by centrifugation to obtain viable cells. 1 ml of 0.1 M potassium phosphate buffer (pH 7) was placed in a screw-mouth test tube, and the above viable cells were added thereto, followed by suspension. To the suspension, 0.1 ml of a raw material (10% (w / v) 4- (methylthio) butane-1,2-diol aqueous solution) (that is, 4- (methylthio) butane-1,2-diol was added). After adding 1% (w / v) as the final concentration, the resulting mixture was shaken at 30 ° C. for 24 hours.
After completion of the reaction, 0.5 ml of the reaction solution was sampled. After removing the cells from the sampling solution, the amount of 2-hydroxy-4-methylthiobutyric acid produced was analyzed by liquid chromatography. The obtained results are shown in Table 1.
(Content analysis conditions)
Column: Cadenza CD-C18 (4.6 mmφ × 15 cm, 3 μm) (manufactured by Imtakt)
Mobile phase: A liquid 0.1% trifluoroacetic acid aqueous solution, B liquid Methanol time (min) A liquid (%): B liquid (%)
0 80:20
10 80:20
20 50:50
30 50:50
30.1 80:20
Flow rate: 0.5 ml / min Column temperature: 40 ° C
Detection: 220nm

Example 2 (Production example of sulfur-containing hydroxycarboxylic acid compound from sulfur-containing dihydroxy compound by the production method of the present invention)
Sterilized medium (1 g of water, 20 g of various alcohols shown in Table 2, 5 g of polypeptone, 3 g of yeast extract, 3 g of meat extract, 0.2 g of ammonium sulfate, 1 g of potassium dihydrogen phosphate and 7 water of magnesium sulfate in a test tube) After adding 0.5 g of the Japanese product, the pH was adjusted to 7.0) 5 ml), and Rhodococcus sp. ATCC19148 strain was inoculated therein. This was cultured with shaking at 30 ° C. under aerobic conditions. After culturing, the cells were separated by centrifugation to obtain viable cells. 1 ml of 0.1 M potassium phosphate buffer (pH 7) was placed in a screw-mouth test tube, and the above viable cells were added thereto, followed by suspension. To the suspension, 0.1 ml of a raw material (10% (w / v) 4- (methylthio) butane-1,2-diol aqueous solution) (that is, 4- (methylthio) butane-1,2-diol was added). After adding 1% (w / v) as the final concentration, the resulting mixture was shaken at 30 ° C. for 24 hours.
After completion of the reaction, 0.5 ml of the reaction solution was sampled. After removing the cells from the sampling solution, the amount of 2-hydroxy-4-methylthiobutyric acid produced was analyzed by liquid chromatography. The obtained results are shown in Table 2.
(Content analysis conditions)
Column: Cadenza CD-C18 (4.6 mmφ × 15 cm, 3 μm) (manufactured by Imtakt)
Mobile phase: A liquid 0.1% trifluoroacetic acid aqueous solution, B liquid Methanol time (min) A liquid (%): B liquid (%)
0 80:20
10 80:20
20 50:50
30 50:50
30.1 80:20
Flow rate: 0.5 ml / min Column temperature: 40 ° C
Detection: 220nm

According to the present invention, a sulfur-containing hydroxycarboxylic acid compound can be produced efficiently.

Claims (4)

Formula (1):

(In the formula, R ¹ represents hydrogen or an alkyl group having 1 to 8 carbon atoms.)
And a treated product of a microorganism belonging to Rhodococcus rhodochrous having the ability to oxidize the sulfur-containing dihydroxy compound to the corresponding α-hydroxycarboxylic acid compound. Formula (2):

(Wherein R ¹ represents the same meaning as described above.)
And the microorganism is allowed to act on the sulfur-containing dihydroxy compound represented by the formula (1) with the microbial cell or the treated product of the microorganism. A production method characterized by being a microorganism cultured in the presence of a primary or secondary alcohol. Formula (1):

(In the formula, R ¹ represents hydrogen or an alkyl group having 1 to 8 carbon atoms.)
Rhodococcus rhodochrous ATCC15610 strain or Rhodococcus sp ATCC19148 strain having the ability to oxidize the sulfur-containing dihydroxy compound to the corresponding α-hydroxycarboxylic acid compound. Formula (2) for causing the microbial cells of the microorganisms or processed microbial cells to act:

(Wherein R ¹ represents the same meaning as described above.)
And the microorganism is allowed to act on the sulfur-containing dihydroxy compound represented by the formula (1) with the microbial cell or the treated product of the microorganism. A production method characterized by being a microorganism cultured in the presence of a primary or secondary alcohol. The production method according to claim 1 or 2, wherein the primary or secondary alcohol is a primary or secondary alcohol having 1 to 5 carbon atoms. The primary or secondary alcohol is methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-methyl-1-propanol or 2,2-dimethyl-1-propanol. Manufacturing method.